Plant as well as bacterial or fungal protoplasts are cells in which the cell wall was partially or completely removed by either mechanical or enzymatic treatment. Since 1961, when enzymatic methods of protoplast isolation from plant tissues were reported, these cell-wall-less cells faced various periods of “popularity”. In 1970s totipotency of plant protoplasts was demonstrated by generating fertile plants from these cells. This led to a “golden age” period during which major methods and techniques for cell preparation, handling and treatments including approaches for DNA uptake were developed. Further expectations were raised with respect to generation of plants with novel properties using genetic manipulation of protoplasts (e.g. nuclear and organelle transformation, or generation of hybrids and cybrids). Protoplasts were used as a versatile system to study plant cell development and physiology, cytodifferentiation, organellogenesis, membrane transport and plant virus function and interaction of viruses with plant cells. Recent advances in genomics, transcriptomics, proteomics and discovery of fluorescent proteins led to “renaissance” of protoplasts in modern science. Despite regular exploitation of protoplasts to study gene and protein function application of protoplasts in high-throughput assays are rather rare. Reasons for this are absence of efficient, practical and economical methods to handle and maintain cell cultures at large scales. Protoplast isolation is now routine from a wide range of plant species. Typically, a protoplast isolation procedure consists of a filtration step to remove large debris after cell wall digestion and one or several centrifugation steps using solutions osmotically and ionically adjusted for a given species to further purify intact and, in special cases, specific cell types, e.g. guard cells, epidermis cells and other cells. Numerous factors, such as different plant material, pre-isolation, isolation and post-isolation physical and chemical requirements and nutrient composition of media used and combination of growth regulators, influence division frequencies of protoplasts and subsequent development of protoplast-derived colonies. Seasonal and internal clock conditions may influence cell behaviour even in vitro, but very likely is species specific.
After preparation cells are typically used for analysis and subsequent culture immediately. Protoplasts can be used, for example, for drug assays, transient and/or stable transformation or somatic hybridisation. However, as freshly prepared plant protoplasts do not easily take up foreign nucleic acids protocols need to be developed for efficient DNA uptake at large scale.
BART et al: PLANT METHODS, vol. 2, page 13 (2006) describes a novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. The protoplasts were transformed with various plasmids using PEG as a transformation agent. For the transformation the DNA was dissolved in a liquid.
YAMADA et al: METHODS IN MOLECULAR BIOLOGY, vol. 643 (2010), pages 33-45 describes protocols for the identification of regulatory protein genes involved in alkaloid biosynthesis using a transient RNAi system. Transformation is carried out using PEG as transformation agent and DNA dissolved in a liquid.
CRAIG et al: PLANT CELL REPORTS, vol. 24, no. 10 (2005), pages 603-611 compares particle bombardment of leaf explants and PEG-mediated transformation of protoplasts. The nucleic acids used for PEG-mediated transformation were dissolved in a liquid.
YOO et al: NATURE PROTOCOLS vol. 2, no. 7 (2007), pages 1565-1572 investigates Arabidopsis mesophyll protoplasts as a cell system for transient gene expression analysis. “DNA-PEG-calcium transfection” using DNA in solution is described.
An Advertising Feature of GenVault Corporation, Carlsbad, Calif., USA [Kansagara et al: NATURE METHODS, vol. 5 (September 2008)] describes dry-state, room-temperature storage of DNA and RNA. The nucleic acids stored in this way, however, cannot be directly used in their dry state. Rather, they first have to be eluted and purified before further use, e.g. in transformation.
In contrast to many human and animal cell cultures (e.g. fibroblasts, pancreatic islet cells, human colon cancer cells and many others) plant protoplasts are an example of non-adhesively growing cells. So far only liquid cultured protoplasts could be used in assays enabling high-throughput analysis. There are several drawbacks of liquid culture. The main one is the impossibility to find the same object/cell for microscopy observation again and again over continuous time periods whenever container with cultured protoplasts should be translocated or moved. This becomes particularly essential if e.g. multiple emission channels are to be compared and analyzed by means of computational tools. Only a switch between 2 channels may lead to microvibrations resulting in cell translocation and thus in a shift between different channels. Another limitation is not-avoidable cell aggregation when cultured in liquid medium over continuous time period. This makes impossible appropriate analysis by e.g. microscopy means. In addition, protoplast populations often consist of more than one cell type by origin, which could additionally be at different developmental states. The cellular heterogeneity and data extrapolation is a problem, and liquid culture does not allow to solve it.
Immobilisation of non-adhesively growing cells is necessary to prevent non-predictable and uncontrolled cell movement, which is not avoidable if cells float freely in the culture medium. Protoplast embedding into semi-solid matrixes allows developing cells to generate microenvironments. Numerous reports demonstrated that immobilisation of plant protoplasts resulted in higher plating efficiencies and optimised cell development. Furthermore, effect of drugs and/or physiologically active compounds can be easily investigated by replacing of incubation/culture media. Immobilised cells or surface growing cells could be subjected to automated microscopy to generate image data suitable for statistical analysis afterwards.
US 2002/173037 A1 describes a method of protoplast culture which comprises mixing protoplasts with alginate solution, placing a CaCl2 solution on a glass microslide, placing a mixture of protoplasts and alginate solution on the glass microslide and immediately covering by a glass coverglass, adding CaCl2 solution in an amount of 70 to 100 μl from the sides of coverglass, sliding down the coverglass towards one side after four to ten minutes and placing it in a petridish containing protoplast culture medium, sealing the petridishes with parafilm and incubating in dark/diffused light at 20 to 27° C., and transferring the extra thin alginate layer with 20-25 celled colonies to regeneration medium for development of culture. This process is rather cumbersome, e.g. the coverglasses have to be handled by forceps (see FIG. 1). Thus, it is not suitable for a high-throughput screening or a fully automated process.
Golds et al: J PLANT PHYSIOL, vol. 140, pages 582-587 established the “thin alginate layer” (TAL) technique, in which protoplasts are enmeshed in an alginate medium and placed in liquid culture medium.
PATI al.: PROTOPLASMA, vol. 226, no. 3-4 (2005), pages 217-221 developed “extra thin alginate films” (ETAF) in order to establish a technique for protoplast culture. The ETAF technique described in this reference requires placing protoplasts on a microscope slide and placing a coverglass on top of the cells. The coverglass is then removed with the help of jeweler's forceps. This technique is not suitable for a high-throughput screening or a fully automated process due to the rather complicated handling involving coverglasses and forceps.
The TAL technique may be suitable for cell tracking, but this will require transfer of the carrier into a plate/container appropriate for microscopy. In addition, this method cannot be used for automation of handling procedures and is based on exclusively man-operated manipulation. Also, the TAL-technique is not suitable for high-throughput analysis since culture of polypropylene grids takes place in liquid environment, in which carriers are swimming, rotating etc. and not in multiwell format. This causes movement of the carrier with embedded protoplasts and without manual adjustments it is impossible to find the same object of interest again. Further, this method is not suitable for multi-well format.
The same criteria apply to the ETAF technique. This method is exclusively man-powered, handling is complicated and not suitable for high-throughput analysis. In addition, non-skilled persons cannot avoid high rates of contamination during manipulation. It requires translocation of the formed film by manual manipulations and cannot be automated, thus omitting high-throughput-oriented assays.
Despite existing procedures to immobilise non-adhesively growing cells such as plant protoplasts, none of them is suited efficiently for both, high-throughput and high-content analysis in combination with high resolution microscopy analysis, such as TIRF (Total Internal Reflection Fluorescence) microscopy. Established procedures result in cell trapping at various focal planes, thus increasing impact of artefacts on data quality while performing image analysis. The present invention provides methods which can be carried out in an automated manner, e.g. in high-throughput analysis, and thus allows successfully to overcome most of the above-mentioned obstacles.
The present invention provides an efficient method for high-throughput single cell analysis using robotic handling and automated microscopy. It was surprisingly found that the use of dried DNA for transforming plant cells resulted in highly reproducible transformation efficiencies which is important for automation of the transformation process. Especially the variation in the co-transformation efficiency was lower as compared to known transformation techniques using DNA dissolved in a liquid (see Example 5). It was further found that the transformation efficiency using the dried DNA was very low unless the cells were sedimented prior to or during the transformation.